Second generation immunotoxins (ITs) containing Fab' or IgG antibody linked via hindered disulfide bonds to deglycosylated ricin A chain (IT-A) are showing promising results in Phase I clinical trials in patients with B cell lymphoma. For these ITs to work optimally in the treatment of minimal disease, their immunogenicity and toxicity must be decreased and potency increased. The aim of this proposal will be to 1) increase the potency of IT-As by adding intracellular routing sequences and/or lipids to the A chain and using bispecific ITs which recruit effector cells as well as deliver A chain, 2) define the immunodominant T cell epitopes on the A chain in order to delete or mutate them to render the A chain less immunogenic, and 3) generate and test ricin A chain mutants in our in vitro vascular leak model to determine whether toxicity and enzymatic activities of the A chain can be mapped to different portions of the molecule. If this should be the case, we will attempt to delete the toxicity by mutagenesis. Based on this information, new third generation ITs will be designed and tested in vitro and in vivo.